sign in sign up
<<
Home , Isoamylase

US 20020081670A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2002/0081670 A1 Bisgard-Frantzen et al. (43) Pub. Date: Jun. 27, 2002

(54) DEBRANCHING (30) Foreign Application Priority Data

(75) Inventors: Henrik Bisgard-Frantzen, Bagsvaerd Jul. 2, 1998 (DK) ...... PA 1998 00868 (DK); Allan Svendsen, Birkerod (DK) Publication Classi?cation Correspondence Address: NOVOZYMES NORTH AMERICA, INC. 5 1 I nt. C]7...... C12P 19/04 ; C12N 9/44; C/O NOVO NORDISK OF NORTH AMERICA, C12N 15/00 INC. (52) US. Cl...... 435/101; 435/210; 435/440 405 LEXINGTON AVENUE, SUITE 6400 NEW YORK, NY 10174 (US) (57) ABSTRACT (73) Assignee: Novozymes A/S, Bagsvaerd (DK) (21) Appl. No.: 09/833,435 The invention relates to a genetically engineered variant of a parent starch debranching , i.e. a or an (22) Filed: Apr. 12, 2001 isamylase, the enZyme variant having an improved thermo stability at a pH in the range of 4-6 compared to the parent Related US. Application Data enZyme and/or an increased activity toWards amylopectin and/or glycogen compared to the parent enZyme, to methods (63) Continuation of application No. 09/346,237, ?led on for producing such starch debranching enZyme variants With Jul. 1, 1999, noW Pat. No. 6,265,197, Which is a improved thermostability and/or altered substrate speci?c non-provisional of provisional application No. ity, and to a method for converting starch to one or more 60/094,353, ?led on Jul. 28, 1998. sugars using at least one such enZyme variant.

Patent Application Publication Jun. 27, 2002 Sheet 4 0f 10 US 2002/0081670 A1

SEQ ID NO 4 = isoamylase from Pseudomonas amyloderamosa SEQ ID NO 7 = isoamylase from Pseudomonas sp. SMPI SEQ ID NO 8 = isoamylase from Favobacterium odoratum SEQ ID NO 9 = isoamylase from Sulfolobus acidocaldarius SEQ ID NO 10 = isoamylase from Sulfolobus solfataricus SEQ ID NO 3 = isoamylase from Rhodothennus marinas SEQ ID NO 11 = isoamylase from Zea mays

1 50

SEQ ID NO 4 ......

SEQ ID NO 7 ......

SEQ ID NO 8 ......

SEQ ID NO 9 ......

SEQ ID NO 10 ......

SEQ ID NO 3 ...... SEQ ID NO 11 RLVTHSTRTH YLIGQSQTNW APSPPLPLPM AQKLPCVSSP RPLLAVPAGR

51 100

SEQ ID NO 4 ...... MKCPK ILAALLGCAV

SEQ ID NO 7 ...... MKCPK ILAALLGCAV

SEQ ID NO 8 ...... MFNKYK QISETDMQRT ILAALL'I'GAL

SEQ ID NO 9 ......

SEQ ID NO 10 ...... MAL

SEQ ID NO 3 ...... MSHSA SEQ ID NO 11 WRAGVRGRPN VAGLGRGRLS LHAAAARPVA EAVQAEEDDD DDDEEVAEER

101 150 SEQ ID NO 4 LAGVPAMPAH AAINSMSLGA SYDAQQANIT FRVYSSQATR IVLYLYSAGY SEQ ID NO 7 LAGVPAMPAH AAINSMSLGA SYDAQQANIT FRVYSSQATR IVLYLYSAGY SEQ ID NO 8 LGA. . . . PAW AAINPNKLGA AYDATKANVT FKVYSSKATR IELYLYSTAT SEQ ID NO 9 . . .MKDRPLK PG.EPYPLGA TWIEEEDGVN FVLFSENATK VELLTYSQTR SEQ ID NO 10 FFR'I‘RDRPLR PG.DPYPLGS NWIEDDDGVN FSLFSENAEK VELLLYSLTN SEQ ID NO 3 QPVTSVQAVW PG.RPYPLGA TW. .DGLGVN FALYSQHAEA VELVLFDHPD SEQ ID NO 11 FALGGACRVL AG.MPAPLGA TALRG. .GVN FAVYSSGASA ASLSLFAPGD

151 200 SEQ ID NO 4 GVQESATYTL SPAGSGVWAV TVPVSSIKAA GITGAVYYGY RAWGPNWPYA SEQ ID NO 7 GVQESATYTL SPAGSGVWAV TVPVSSIKAA GITGAVYYGY RAWGPNWPYA SEQ ID NO 8 GSAEKAKYVM TNSG.GIWSV TIPTS'I’LSGQ GLGGTLYYGY RAWGPNWPYN SEQ ID NO 9 QDEPKEIIEL R. . . . .QR'I'G DLWHVFVPGL R.PGQL.YGY RVYGPYKP. . SEQ ID NO 10 QKYPKEIIEV K. . . . .NKTG DIWHVFVPGL R.PGQL.YAY RVYGPYKP. . SEQ ID NO 3 DPAPSRTIEV T. . . . .ERTG PIWHVYLPGL R.PGQL.YGY RVYGPYRP. . SEQ ID NO 11 LKADRVTEEV PLDPLLNRTG NVWHVFIHGD ELHGML.CGY RFDGVFAP. .

Fig. 2(a)

Patent Application Publication Jun. 27, 2002 Sheet 8 0f 10 US 2002/0081670 A1

SEQ ID NO 1 = pullulanase from Bacillus acidopullulyticus SEQ ID NO 2 = pullulanase from Bacillus deramificans SEQ ID NO 3 = isoamylase from Rhodothermus marinus SEQ ID NO 4 = isoamylase from Pseudomonas amyloderamosa

5O Seq. No.1 VSLIRSRYNI-I FVILFTVAIM ALADSTSTEV IVHYHRFDSN

Seq .No.2 ...... u u - u . e n n - I . . .DGNTTTI IVHYFRPAGD

Seq .No.3 ......

Seq .No.4 ...... - - - - e - - - - -

51 100 Seq. No.1 YANWDLWMWP YQPVNGNGAA YEFSGKDD.F GVKADVQVPG DDTQVGLIVR Seq. No.2 YQPWSLWMW. . .PKDGGGAE YDFNQPADSF GAVASADI PG NPSQVGIIVR

Seq. No.3 ...... e - - . , - - I ------. . - - -

Seq . No.4 ...... - . . . I . . . -

101 150 Seq .No.1 TNDWSQKNTS DDLHIDLTKG HEIWIVQGDP NIYYNLSDAQ AAATPKVSNA Seq .No.2 TQDWT.KDVS ADRYIDLSKG NEVWLVEGNS QIFYNEKDAE DAAKPAVSNA

Seq. No.3 ......

Seq. No.4 ......

151 200 Seq .No.1 YLDNEKTVLA KLTNPMTLSD GSSGFTVTDK TTGEQIPVTA ATNANSA. . . Seq. No.2 YLDASNQVLV KLSQPLTLGE GASGFTVHDD TANKDIPVTS VKDASLGQDV

Seq. No.3 ...... ~ - - - - - . - -

Seq. No.4 ...... - - - - ¢ - . - - -

201

Seq. No. 1 ...... - Q - I | - . - - ~ e . u I - | u - - I - l . u u . 1 . . . - u . . . - . - . Seq. No.2 TAVLAGTFQH

Seq. No.3 ...... - . . . I . . . . - . . v . - - - . -

Seq .No.4 ......

251

Seq. No. 1 ...... Q - - v - e u - I u - o n e - l I I o . 0 l - l - l - . l - Seq. No.2 LNDSWNNPSY PSDNINLTVP AGGAHVTFSY IPSTHAVYDT INNPNADLQV

Seq .No.3 ...... MSHSAQPVT SVQAVWPGRP YPLGATWDGL GVNFAL. .YS Seq. No.4 MKCPKILAAL LGCAVLAGVP AMPAHAAINS MSLGASYDAQ QANITFRVYS

301 350 Seq. No.1 SSSEQTDLVQ LTLASAPDVS HTIQVGAAGY EAVNLIPRNV LNLPRYYYSG Seq. No.2 ESGVKTDLVT VTLGEDPDVS HTLSIQTDGY QAKQVIPRN'V LNSSQYYYSG

Seq .No.3 QHAEAVELVL FDHPDDPAPS RTIEV'I'ER'I‘G PIWHVY. . . . LP ...... G Seq. No.4 SQATRIVLYL YSAGYGVQES ATYTLSPAGS GVWAVT. . . . VPVSSIKAAG Fig. 3(a)

US 2002/0081670 A1 Jun. 27, 2002

STARCH DEBRANCHING ENZYMES “lique?ed” so that it can be handled. This reduction in viscosity is today mostly obtained by enZymatic degrada CROSS-REFERENCE TO RELATED tion. APPLICATIONS [0010] Liquefaction [0001] This application is a continuation of US. applica tion Ser. No. 09/346,237, ?led Jul. 1, 1999, Which claims [0011] During the liquefaction step, the long-chained priority under 35 U.S.C. 119 of Danish application PA 1998 starch is degraded into smaller branched and linear units 00868, ?led Jul. 2, 1998, and the bene?t of US. provisional (maltodextrins) by an ot- (e.g. TermamylTM, avail application Ser. No. 60/094,353 ?led on Jul. 28, 1998, the able from Novo Nordisk A/S, Denmark). The liquefaction contents of Which are fully incorporated herein by reference. process is typically carried out at about 105-110° C. for about 5 to 10 minutes folloWed by about 1-2 hours at about FIELD OF THE INVENTION 95° C. The pH generally lies betWeen about 5.5 and 6.2. In order to ensure an optimal enZyme stability under these [0002] The present invention relates to novel starch conditions, calcium is added, eg 1 mM of calcium (40 ppm debranching enZymes, in particular and free calcium ions). After this treatment the lique?ed starch isoamylases, designed for use in a starch conversion process Will have a “dextrose equivalent” (DE) of 10-15. comprising a liquefaction step and a sacchari?cation step, as Well as to the production of such enZymes and the use of [0012] Sacchari?cation such enZymes in a starch conversion process. [0013] After the liquefaction process the maltodextrins are BACKGROUND OF THE INVENTION converted into dextrose by addition of a glucoamylase (e.g. AMGTM, available from Novo Nordisk A/S) and a debranch [0003] such as corn, potato, Wheat, manioc and ing enZyme, such as an isoamylase (see eg U.S. Pat. No. rice starch are used as the starting material in commercial 4,335,208) or a pullulanase (e.g. PromoZymeTM, available large scale production of sugars, such as high fructose syrup, from Novo Nordisk A/S) (see U.S. Pat. No. 4,560,651). high maltose syrup, maltodextrins, amylose, G4-G6 oli Before this step the pH is reduced to a value beloW 4.5, eg gosaccharides and other carbohydrate products such as fat about 3.8, maintaining the high temperature (above 950 C.) replacers. for a period of eg about 30 min. to inactivate the liquefying ot-amylase to reduce the formation of short oligosaccharides [0004] Degradation of starch called “panose precursors” Which cannot be hydrolyZed [0005] Starch usually consists of about 80% amylopectin properly by the debranching enZyme. and 20% amylose. Amylopectin is a branched polysaccha ride in Which linear chains ot-1,4 D-glucose residues are [0014] The temperature is then loWered to 60° C., glu joined by ot-1,6 glucosidic linkages. Amylopectin is partially coamylase and debranching enZyme are added, and the degraded by ot-amylase, Which hydrolyZes the 1,4-ot-gluco sacchari?cation process proceeds for about 24-72 hours. sidic linkages to produce branched and linear oligosaccha [0015] Normally, When denaturing the ot-amylase after the rides. Prolonged degradation of amylopectin by ot-amylase liquefaction step, a small amount of the product comprises results in the formation of so-called ot-limit dextrins Which panose precursurs Which cannot be degraded by pullulanases are not susceptible to further by the ot-amylase. or AMG. If active amylase from the liquefaction step is Branched oligosaccharides can be hydrolyZed into linear present during sacchari?cation (i.e. no denaturing), this level oligosaccharides by a debranching enZyme. The remaining can be as high as 1-2% or even higher, Which is highly branched oligosaccharides can be depolymeriZed to D-glu undesirable as it loWers the sacchari?cation yield signi? cose by glucoamylase, Which hydrolyZes linear oligosaccha cantly. For this reason, it is also preferred that the ot-amylase rides into D-glucose. is one Which is capable of degrading the starch molecules [0006] Amylose is a linear polysaccharide built up of into long, branched oligosaccharides (such as, e.g., the D-glucopyranose units linked together by ot-1,4 glucosidic FungamylTM-like ot-) rather than shorter branched linkages. Amylose is degraded into shorter linear oligosac oligosaccharides. charides by ot-amylase, the linear oligosaccharides being depolymeriZed into D-glucose by glucoamylase. [0016] IsomeriZation [0007] In the case of converting starch into a sugar, the [0017] When the desired ?nal sugar product is eg high starch is depolymeriZed. The depolymeriZation process con fructose syrup, the dextrose syrup may be converted into sists of a pretreatment step and tWo or three consecutive fructose by enZymatic isomeriZation. After the sacchari?ca process steps, namely a liquefaction process, a sacchari? tion process the pH is increased to a value in the range of cation process and, depending on the desired end product, 6-8, preferably about pH 7.5, and the calcium is removed by optionally an isomeriZation process. ion exchange. The dextrose syrup is then converted into high fructose syrup using, e.g., an immobiliZed glucose [0008] Pre-treatment of native starch (such as SWeetZymeTM, available from Novo Nordisk A/S). [0009] Native starch consists of microscopic granules [0018] Debranching enZymes Which are insoluble in Water at room temperature. When an aqueous starch slurry is heated, the granules sWell and [0019] Debranching enZymes Which can attack amylopec eventually burst, dispersing the starch molecules into the tin are divided into tWo classes: isoamylases (EC. 3.2.1.68) solution. During this “gelatiniZation” process there is a and pullulanases (EC. 3.2.1.41), respectively. Isoamylase dramatic increase in viscosity. As the solids level is 30-40% hydrolyses ot-1,6-D-glucosidic branch linkages in amy in a typical industrial process, the starch has to be thinned or lopectin and [3-limit dextrins and can be distinguished from US 2002/0081670 A1 Jun. 27, 2002

pullulanases by the inability of isoamylase to attack pullu [0028] identifying one or more homologous amino lan, and by their limited action on ot-limit deXtrins. acid residues and/or amino acid regions in a second parent starch debranching enZyme by means of [0020] When an acidic stabilised “TermamylTM-like”ot alignment of the amino acid sequences of the ?rst amylase is used for the purpose of maintaining the amylase and second parent starch debranching enZymes, and activity during the entire sacchari?cation process (no inac tivation), the degradation speci?city should be taken into [0029] mutating one or more of the homologous consideration. It is desirable in this regard to maintain the amino acid residues and/or amino acid regions in the ot-amylase activity throughout the sacchari?cation process, second parent starch debranching enZyme to produce since this alloWs a reduction in the amyloglucidase addition, an enZyme variant With increased thermostability. Which is economically bene?cial and reduces the AMGTM [0030] A still further aspect of the invention relates to a condensation product isomaltose, thereby increasing the DE method for producing a starch debranching enZyme variant (dextrose equivalent) yield. With altered substrate speci?city, the method comprising the [0021] It Will be apparent from the above discussion that steps of: the knoWn starch conversion processes are performed in a [0031] identifying one or more amino acid residues in series of steps, due to the different requirements of the at least one amino acid loop associated With speci various enZymes in terms of eg temperature and pH. It ?city toWards a desired substrate in a ?rst parent Would therefore be desirable to be able to engineer one or starch debranching enZyme, more of these enZymes so that the overall process could be performed in a more economical and ef?cient manner. One [0032] identifying one or more homologous amino possibility in this regard is to engineer the otherWise ther acid residues in at least one corresponding loop in a molabile debranching enZymes so as to render them more second parent starch debranching enZyme by means stable at higher temperatures. The present invention relates of alignment of the amino acid sequences of the ?rst to such thermostable debranching enZymes, the use of Which and second parent starch debranching enZymes, and provides a number of important advantages Which Will be [0033] mutating one or more of the homologous discussed in detail beloW. It also relates to starch debranch amino acid residues in at least one loop in the second ing enZymes With an altered substrate speci?city. parent starch debranching enZyme to produce an enZyme variant With altered substrate speci?city. SUMMARY OF THE INVENTION [0034] The term “loop” means, at least in the context of [0022] An object of the present invention is thus to pro the present invention, the sequence part folloWing the beta vide thermostable debranching enZymes, for eXample pul strand/sheet part of the sequence in question. Said “beta lulanases and isoamylases, Which are suitable for use at high strands/sheets” may be identi?ed by multiple sequence temperatures in a starch conversion process, in particular alignment of sequences of the present invention and using genetic engineering techniques in order to identify and sequences With a knoWn three dimensional structure. Such synthesiZe suitable enZyme variants. Another object of the alignments can be made using standard alignment programs, invention is to provide novel starch debranching enZymes available from eg the UWGCG package (Program Manual With an altered substrate speci?city. for the Wisconsin Package, Version 8, August 1994, Genet ics Computer Group, 575 Science Drive, Madison, Wis., [0023] In its broadest aspect, the present invention can USA 53711). thus be characteriZed as relating to novel starch debranching enZymes With improved properties in terms of eg thermo [0035] KnoWn three-dimensional enZyme structures are stability or substrate speci?city, as Well as methods for available from Brookhaven Databank. EXamples of such are producing such enZymes and the use of such enZymes in a the three-dimensional structure of the Aspergillus oryZae starch conversion process. TAKA ot-amylase (SWift et al., 1991), the Aspergillus niger acid amylase (Brady et al, 1991), the structure of pig [0024] In one particular aspect, the invention relates to a pancreatic ot-amylase (Qian et al., 1993), and the barley genetically engineered variant of a parent starch debranch ot-amylase (KadZiola et al. 1994, Journal of Molecular ing enZyme, the enZyme variant having an improved ther Biology 239:104-121; A.KadZiola, Thesis, Dept of Chem mostability at a pH in the range of 4-6 compared to the istry, U. of Copenhagen, Denmark). parent enZyme. [0036] The invention relates in addition to a method for [0025] Another aspect of the invention relates to a geneti converting starch to one or more sugars, the method com cally engineered variant of a parent starch debranching prising debranching the starch using at least one enZyme enZyme, the enZyme variant having an increased activity variant as described herein. toWards amylopectin and/or glycogen compared to the par ent enZyme. DETAILED DESCRIPTION OF THE INVENTION [0026] A further aspect of the invention relates to a method for producing a starch debranching enZyme variant [0037] In the present conteXt, the term “thermostable” With increased thermostability, the method comprising the refers in general to the fact that the debranching enZyme steps of: variants according to the invention have an improved ther mostability compared to the relevant parent enZyme. The [0027] identifying one or more amino acid residues degree of improvement in thermostability can vary accord and/or amino acid regions associated With thermo ing to factors such as the thermostability of the parent stability in a ?rst parent starch debranching enZyme, enZyme and the intended use of the enZyme variant, i.e. US 2002/0081670 A1 Jun. 27, 2002

Whether it is primarily intended to be used in for liquefaction activity of the parent enZyme under the given set of condi or for sacchari?cation or both. It Will be apparent from the tions mentioned in connection With improved thermostabil discussion below that for sacchari?cation, the enZyme vari ity right above. ant should maintain a substantial degree of enZyme activity during the sacchari?cation step at a temperature of at least [0045] An enZyme variant “derived from” a given enZyme about 63° C., preferably at least about 70° C., While an (a “parent enzyme”) means that the amino acid sequence of enZyme variant designed for use in the liquefaction step the parent enZyme has been modi?ed, ie by substitution, should be able to maintain a substantial degree of enZyme deletion, insertion and/or loop transfer as described beloW, activity at a temperature of at least about 95° C. to result in the enZyme variant. In the case of a parent [0038] The improved thermostability of enZyme variants enZyme produced by an organism such as a microorganism, according to the invention can in particular be de?ned Where an enZyme variant according to the invention is according to one or more of the folloWing criteria: derived from the parent enZyme, the enZyme variant may be produced by appropriate transformation of the same or a [0039] In one embodiment, the enZyme variant of the similar microorganism or other organism used to produce invention has an improved thermostability as de?ned by the parent enZyme. differential scanning calorimetry (DSC) using the method described beloW. [0046] One advantage of the thermostable debranching enZymes of the invention is that they make it possible to [0040] In another embodiment, the enZyme variant of the perform liquefaction and debranching simultaneously before invention has an improved thermostability as de?ned by an the sacchari?cation step. This has not previously been increased half-time (T,/2) of at least about 5%, preferably at possible, since the knoWn pullulanases and isoamylases With least about 10%, more preferably at least about 15%, more acceptable speci?c activity are thermolabile and are inacti preferably at least about 25%, most preferably at least about vated at temperatures above 60° C. (Some thermostable 50%, such as at least 100%, in the “Tl/2 assay for liquefac pullulanases from Pyrococcus are knoWn, but these have an tion” described herein, using a pH of 5.0 and a temperature extremely loW speci?c activity at higher temperatures and of 95° C. EnZyme variants according to this de?nition are are thus unsuitable for purposes of the present invention). By suitable for use in the liquefaction step of the starch con debranching, using the thermostable debranching enZymes version process. of the invention, during liquefaction together With the action [0041] Alternatively or additionally, an enZyme variant of an ot-amylase, the formation of panose precursors is suitable for use in liquefaction can be de?ned as having an reduced, thereby reducing the panose content in the ?nal improved thermostability as de?ned by an increased residual product and increasing the overall sacchari?cation yield. It enZyme activity of at least about 5%, preferably at least is also possible in this manner to eXtend the liquefaction about 10%, more preferably at least about 15%, more process time Without risking formation of large amount of preferably at least about 25%, most preferably at least about panose precursors. By prolonging the liquefaction step, the 50%, in the “assay for residual activity after liquefaction” DE yield is increased from 10-15 to eg 15-20, reducing the described herein, using a pH of 5.0 and a temperature of 95° need for glucoamylase. This reduced glucoamylase require C. ment is in turn advantageous as the formation of undesired isomaltose is reduced, thereby resulting in an increased [0042] In a further embodiment, the enZyme variant of the glucose yield. In addition, the reduced glucoamylase addi invention has an improved thermostability as de?ned by an tion enables the sacchari?cation step to be carried out at a increased half-time (Tl/2) of at least about 5%, preferably at higher substrate concentration (higher DS, dry substances, least about 10%, more preferably at least about 15%, more concentration) than the normal approx. 30-35% used accord preferably at least about 25%, most preferably at least about ing to the prior art. This alloWs reduced evaporation costs 50%, such as at least 100%, in the “Tl/2 assay for sacchari doWnstream, eg in a high fructose corn syrup process, and ?cation” described herein, using a pH of 4.5 and a tempera the sacchari?cation reaction time can also be reduced, ture of 70° C. Such variants are suitable for use in the thereby increasing production capacity. A further advantage sacchari?cation step of the starch conversion process. is that ot-amylase used in the liquefaction process does not [0043] Alternatively or additionally, an enZyme variant need to be inactivated/denatured in this case. suitable for sacchari?cation can be de?ned as having an improved thermostability as de?ned by an increased residual [0047] Furthermore, it is also possible to use the thermo enZyme activity of at least about 5%, preferably at least stable debranching enZymes according to the invention about 10%, more preferably at least about 15%, more during sacchari?cation, Which is advantageous for several preferably at least about 25%, most preferably at least about reasons. In the conventional starch sacchari?cation process, 50%, in the “assay for residual activity after sacchari?ca the process temperature is not more than 60° C. due to the fact that neither the sacchari?cation enZyme pullulanase nor tion” described herein, using a pH of 4.5 and a temperature of 63° C. Preferably, this improved thermostability is also AMGTM are suf?ciently thermostable to alloW the use of a observed When assayed at a temperature of 70° C. higher temperature. This is a disadvantage, hoWever, as it Would be very desirable to run the process at a temperature [0044] The term “substantially active” as used herein for of above about 60° C., in particular above 63° C., eg about a given enZyme variant and a given set of conditions of 70° C., to reduce microbial groWth during the relatively long temperature, pH and time means that the relative enZymatic sacchari?cation step. Furthermore, a higher process tem activity of the enZyme variant is at least about 25%, pref perature normally gives a higher activity per mg of enZyme erably at least about 50%, in particular at least about 60%, (higher speci?c activity), thereby making it possible to especially at least about 70%, such as at least about 90% or reduce the Weight amount of enZyme used and/or obtain a 95%, eg at least about 99% compared to the relative higher total enZymatic activity. A higher temperature can US 2002/0081670 A1 Jun. 27, 2002

also result in a higher dry matter content after sacchari?ca [0056] Invert the test tube and shake vigorously. Measure tion, Which Would be bene?cial in terms of reducing evapo the absorbance at 520 nm, inverting the test tube once ration costs. immediately prior to transfer of the liquid to the cuvette. [0048] Although a thermostable isoamylase might be [0057] Blank value: Same procedure as for the sample regarded as being more bene?cial than a thermostable value, but With Water instead of sugar solution. pullulanase When used in the liquefaction process, since isoamylases are characterised by their speci?city toWards [0058] Standard value: Same procedure as for the sample amylopectin and activity on higher molecular Weight dex value. trins, a preferred alternative is to alter the speci?city of a [0059] Calculations: pullulanase so as to be more “isoamylase-like” in the sense of having improved activity toWards longer, branched-chain [0060] In the region 0-2 the absorbance is proportional to dextrins. Among the various pullulanases there are substan the amount of sugar. tial differences in this respect, even among the pullanases of the same Bacillus origin. 100 (sample — blank) mg sugar/l : [0049] Methods for determining stability and activity (standard — blank) (sample — blank) [0050] Thermostability % glucose : [0051] Thermostability of pullulanases and isoamylases 100 X (standard — blank) can be detected by measuring the residual activity by incubating the enZyme under accelerated stress conditions, [0061] Reagents: Which comprise: pH 4.5 in a 50 mM sodium acetate buffer Without a stabiliZing dextrin matrix (such as the approxi [0062] 1. Somogyi’s copper reagent mately 35% dry matter Which is normally present during [0063] 35.1 g Na2HPO4.2H2O and 40.0 g potassium sacchari?cation). The stability can be determined at iso sodium tartrate (KNaC4H4O2.4H2O) are dissolved in 700 ml therms of eg 63° C., 70° C., 80° C., 90° C. and 95° C., of deioniZed Water. 100 ml of 1N sodium hydroxide and 80 measuring the residual activity of samples taken from a ml of 10% cupric sulphate (CuSO4.5H2O) are added. 180 g Water bath at regular intervals (e.g. every 5 or 10 min.) of anhydrous sodium sulphate are dissolved in the mixture, during a time period of 1 hour. For determining stability for and the volume is brought to 1 liter With deioniZed Water. the purpose of liquefaction, a pH of 5 .0, a temperature of 95° C. and a total assay time of 30 minutes are used (“assay for [0064] 2. Nelson’s colour reagent residual activity after liquefaction”). For determining stabil [0065] 50 g of ammonium molybdate are dissolved in 900 ity for the purpose of sacchari?cation, a pH of 4.5, a ml of deioniZed Water. Then 42 ml of concentrated sulphuric temperature of 63° C. or 70° C. and a total assay time of 30 acid are added, folloWed by 6 g of disodium hydrogen minutes are used (“assay for residual activity after saccha arsenate heptahydrate dissolved in 50 ml of deioniZed Water, ri?cation”). and the volume is brought to 1 liter With deioniZed Water. [0052] Alternatively, the thermostability may be The solution is alloWed to stand for 24-48 hours at 37° C. expressed as a “half-time” (Tl/2), Which is de?ned as the before use and is stored in the dark in a broWn glass bottle time, under a given set of conditions, at Which the activity With a glass stopper. of the enZyme being assayed is reduced to 50% of the initial activity at the beginning of the assay. In this case, the “Tl/2 [0066] 3. Standard assay for liquefaction” uses a pH of 5.0 and a temperature [0067] 100 mg of glucose (anhydrous) are dissolved in 1 of 95° C., While the “Tl/2 assay for sacchari?cation” uses a liter of deioniZed Water. pH of 4.5 and a temperature of 70° C. The assay is otherWise performed as described above for the respective assays for [0068] Substrate speci?city residual activity. [0069] Methods for the determination and characterisation [0053] Activity: Somogyi—Nelson method for determina of the pro?le of action and speci?city of pullulanases and tion of reducing sugars isoamylases for various substrates (e.g. amylopectin, glyco gen and pullulan) are described by Kainuma et al. in [0054] The activity of both pullulanases and isoamylases Carbohydrate Research, 61 (1978) 345-357. Using these can be measured using the Somogyi—Nelson method for the methods, the relative activity of an isoamylase or a pullu determination of reducing sugars (J. Biol. Chem. 153, 375 lanase can be determined, and the relative activity of an (1944)). This method is based on the principle that sugar enZyme variant according to the invention compared to the reduces cupric ions to cuprous oxide, Which reacts With an relative activity of the parent enZyme can be assessed, for arsenate molybdate reagent to produce a blue colour that is example to determine Whether a pullulanase variant has the measured spectrophotometrically. The solution to be mea desired increased speci?city toWard high molecular Weight sured must contain 50-600 mg of glucose per liter. The saccharides such as amylopectin compared to the parent procedure for the Somogyi—Nelson method is as folloWs: enzyme. [0055] Sample value: Pipet 1 ml of sugar solution into a [0070] Starch conversion test tube. Add 1 ml of copper reagent. Stopper the test tube With a glass bead. Place the test tube in a boiling Water bath [0071] As indicated above, in one embodiment of the for 20 minutes. Cool the test tube. Add 1 ml of Nelson’s invention, the starch conversion process comprises colour reagent. Shake the test tube Without inverting it. Add debranching using a thermostable debranching enZyme of 10 ml of deioniZed Water. the invention during the liquefaction step together With an US 2002/0081670 A1 Jun. 27, 2002

ot-amylase. The liquefaction step is typically carried out at 293:781-788, 1993). This suggests that they have the same a pH between 4.5 and 6.5, eg from 5.0 to 6.2, at a overall structure in the central part of the molecule consist temperature in the range of 95-110° C. for a period of 1 to ing of an A, B and C domain. The B domains vary quite 3 hours, preferably about 1.5-2 hours. It is preferred, hoW dramatically in siZe and structure, Whereas the other tWo ever, that the pH is as loW as possible, eg from 4.5 to 5.0, domains are believed to generally possess a high degree of as long as the enZyme(s) used for the liquefaction have a homology. The A domain is composed of an alpha-8/beta-8 sufficient stability at the pH in question. If the ot-amylase is structure (a beta-barrel) and 8 loops betWeen the beta calcium dependent, calcium may be added in an amount of strands and the alpha-helices (a helix can in certain cases be from 30 to 50 ppm, such as around 40 ppm (or 0.75 to 1.25 absent, hoWever). The sequences coming from the beta mM, such as around 1 mM) in the liquefaction step to strand part of the beta-barrel point toWards the substrate stabilise the enZyme. As explained above, the the ot-amylase binding region. These regions are of particular interest for need not be inactivated after the liquefaction step to reduced the speci?city of the enZyme (Svensson, B. et al., Biochemi the panose formation in this case. cal Society Transactions, Vol. 20; McGregor, J. Prat. Chem. [0072] Examples of speci?c ot-amylases Which can be 7:399, 1988). HoWever, by using information about speci?c used in the liquefaction step include Bacillus licheniformis sequences and as Well as general strategies for analyZing ot-amylases, such as the commercially available products pullulanase and isoamylase sequences, the present invention Termamyl®, SpeZyme® AA, SpeZyme® Delta AA, Max provides the necessary tools to be able to engineer these amyl® and Kleistase®, and the ot-amylase mutants enZymes to produce variants With improved thermostability described in WO 96/23874 (Novo Nordisk) and PCT/DK97/ and/or speci?city. 00197 (Novo Nordisk). [0080] Sequence listings [0073] Isoamylases Which can be used as a parent enZyme [0081] The folloWing sequence listings are referred to according to the invention include, but are not limited to, the herein: thermostable isoamylase derived from the thermophilic acrhaebacterium Sulfolobus acidocala'arius (Maruta, K et [0082] SEQ ID NO 1: pullulanase from Bacillus acidop al., (1996), Biochimica et Biophysica Acta 1291, p. 177 ullulyticus 181), isoamylase from Rhodothermus marinus (eg the isoamylase of SEQ ID NO 3) and isoamylase from [0083] SEQ ID NO 2: pullulanase from Bacillus derami Pseudomonas, e.g. Pseudomonas amyloderamosa (e.g. ?cans Pseudomonas amyloderamosa isoamylase disclosed in [0084] SEQ ID NO 3+SEQ ID NO 12: isoamylase from EMBL database accession number J03871 or GeneBank Rhodothermus marinus accession number N90389). [0074] Examples of pullulanases Which can be used as a [0085] SEQ ID NO 4: isoamylase from Pseudomonas parent enZyme include, but are not limited to, a thermostable amyloderamosa JD270 (Chen,] H et al. (1990) Biochemica pullulanase from eg Pyrococcus or a protein engineered et Biophysica Acta 1087, pp 307-315) (Brookhaven data pullulanase from eg a Bacillus strain such as Bacillus base: 1BF2) acidopullulyticus (e.g. PromoZymeTM or SEQ ID NO 1) or [0086] SEQ ID NO 5: pullulanase from Klebsiella pneu Bacillus derami?cans (e.g. SEQ ID NO 2; or the Bacillus moniae (Kornacker et al., Mol. Microbiol. 4:73-85(1990)) derami?cans pullulanase With GeneBank accession number Q68699). [0087] SEQ ID NO 6: pullulanase from Klebsiella aero genes (Katsuragi et al., J. Bacteriol. 169:2301-2306 (1987)) [0075] While prior art methods for sacchari?cation employ a temperature of not more than about 60° C., the [0088] SEQ ID NO 7:isoamylase from Pseudomonas sp. present invention provides thermostable debranching SMP1 (Tognoni,A. et al., US. Pat. No. 5,457,037) enZymes that can remain active at higher temperatures, ie at least about 63° C. and preferably at least about 70° C. so [0089] SEQ ID NO 8: isoamylase from Favobacterium as to eliminate possibilities for microbial groWth. Examples odoratum (JP 9623981, Susumu HiZukuri et al.) of suitable glycoamylases for sacchari?cation include [0090] SEQ ID NO 9: isoamylase from Sulfolobus aci Aspergillus niger glucoamylases, such as AMGTM. The docala'arius, ATCC 33909 (Biochimica et Biophysica Acta sacchari?cation process typically proceeds for about 24-72 1291 (1996) 177-181, KaZuhiko Maruta et al.) hours at a pH of about 4.0-4.5, preferably about 4.0. [0076] When the desired ?nal sugar product is eg a high [0091] SEQ ID NO 10: isoamylase from Sulfolobus s0l fructose syrup of approx. 50% fructose syrup, the formed fataricus (GeneBank Accession no. Y08256). D-glucose is isomeriZed by an isomerase at a pH around 6-8, [0092] SEQ ID NO 11: isoamylase from maiZe, Zea mays preferably about 7.5. An example of a suitable isomerase is (ACCESSION U18908) an glucose isomerase such as the glucose isomerase derived from Streptomyces murinus. The isomerase may be an [0093] SEQ ID NO 13: Bacillus acidopullulyticus pulB immobiliZed glucose isomerase, such as SWeetZyme®. gene (SEQ ID NO: 13). [0077] Calcium is normally removed if added before the [0094] Structure of pullulanases liquefaction step. [0095] The appended FIG. 1 shoWs the amino acid [0078] Engineering of pullulanases and isoamylases sequence of four different pullulanases as Well as an align [0079] The pullulanases and isoamylases are members of ment of these sequences. On the basis of information pro the family 13 amylases (Henrissat, B. et al., Biochem J. vided by this alignment, it is possible to perform homology US 2002/0081670 A1 Jun. 27, 2002

substitutions in order to obtain desired characteristics of number, indicating the position in the sequence of the loops. improved thermostability and/or altered substrate speci?c Information on the location of the individual loops is found ity. in Table 1 beloW. [0096] The four sequences are SEQ ID NO 5, 6, 1 and 2. [0103] By comparing the most homologous sequence [0097] Structure of isoamylases from the “key alignment” of tWo or more relevant starch debranching enZymes With a neW starch debranching [0098] The appended FIG. 2 shoWs the amino acid enZyme sequence, and aligning these tWo sequences, resi sequence of seven different isoamylases as Well as an dues from the neW sequence homologous to residues in the alignment of these sequences. On the basis of information sequence from the key alignment can be determined. The provided by this alignment, it is possible to perform homol homology may be found eg by using the GAP program ogy substitutions in order to obtain desired characteristics of from the UWGCG package (Program Manual for the Wis improved thermostability and/or altered substrate speci?c consin Package, Version 8, August 1994, Genetics Computer 1ty. Group, 575 Science Drive, Madison, Wis., USA 53711). The [0099] The seven sequences are SEQ ID NO 4, 7, 8, 9, 10, neW sequence can then be placed in the key alignment by 3 and 11. using a teXt editing program or other suitable computer [0100] The X-ray structure of the Pseudomonas amy program. loderamosa isoamylase has recently been published in the [0104] Table 1 beloW provides information on the location Brookhaven database under number 1BF2. The structure of selected regions of interest in the various loops of the con?rms the overall vieW of the sequence alignment selected pullulanases and isoamylases, these loop regions method, but also shoWs certain differences to the suggested being of general interest With regard to modi?cation to alignment. The corrected loop numbers deduced from the produce enZyme variants according to the invention. Loop 3 3D structure of Pseudomonas amyloderamosa isoamylase beloW constitutes domain B (MacGregor, 1988), While the (1BF2) are shoWn beloW: other loops belong to domain A.

TABLE 1

Loop 1 Loop 2 Loop 3 Loop 4 Loop 5 Loop 6 Loop 7 Loop 8

Pullulanases

Seg. Id. 21. 369-397 419-458 484-525 553-568 582-608 613-616 661-670 708-739 No. 1 b. 372-385 422-448 488-520 558-568 587-606 663-670 710-724 c. 375-385 422-438 488-499 591-606 710-715 Seq. Id. 21. 465-493 515-554 580-621 649-684 680-711 714-717 757-765 804-834 No. 2 Isoamylases

Seq. Id. 21. 210-230 250-292 319-376 401-437 461-479 482-485 530-539 605-636 No. 4 Seq. Id. 21. 176-195 218-257 283-334 359-381 395-413 416-419 461-470 537-568 No. 3 b. 179-193 221-250 288-326 364-381 399-411 463-470 539-553 c. 182-193 221-239 288-303 403-411 539-545

[0105] Where more than one region is listed for a given enZyme and loop in Table 1, the region listed ?rst (i.e. a.) is in each case the eXpected length of the loop in question. The LOOP Suggested Deduced from Structure neXt region (i.e. b.) is the preferred region for modi?cation, and the last region (i.e. c.) is most preferred. 1. 210-230 211-231 2' 250-292 251-295 [0106] By performing modi?cations, i.e. substitutions, 3. 319-376 319-330 . . . , 4 401 436 401436 deletions, Insertions and/or loop transfer, In one or more 5' 461_479 461468 these loops, engineered proteins having the desired proper 6_ 482485 482403 ties in terms ‘of ‘improved thermostability and/or altered 7_ 53O_539 533_580 substrate speci?city may be produced. An enZyme variant 8. 605-636 606-636 according to the invention may comprise any appropriate combination of one or more substitutions, deletions, inser tions and/or loop transfers to obtain the desired character [0101] Alignment of pullulanases and isoamylases istics of improved thermostability and/or altered activity. [0107] The loop region of SEQ ID NO: 1, i.e. 369-397 [0102] The appended FIG. 3 shoWs a “key alignment” of (region denoted a), may according to the invention suitably selected pullulanases and isoamylases (those of SEQ ID NO be replaced With the corresponding spatially placed region 1, 2, 3 and 4), in other Words a best-?t alignment of of SEQ ID NO.4, i.e. 176-195 (i.e. denoted a.). Further, homologous amino acid residues in the respective region 371-385 of loop 1 (SEQ ID NO: 1) (i.e. denoted b.) sequences. The dashes (“----”) indicate presumed beta may correspondingly be replaced With region 179-193 of strand positions. Each set of dashes is folloWed by a loop loop 1 (SEQ ID NO. 4) (i.e. denoted b.). US 2002/0081670 A1 Jun. 27, 2002

[0108] In the present context, a simpli?ed nomenclature is [0117] Positions for proline substitution include G81P, used to indicate amino acid substitutions in a given position. G99P, T18P, T199P, Q202P, T221, Q226P, A238P, T278P, For example “G81P” refers to the substitution of a glycine R286P, A294P, G467P, G64P, V67P, E69P, A549P, G713P, residue in position 81 With a proline residue, and “F489G,A” T719P and D736P, and preferably S356P, T376P, T278P, refers to the substitution of a phenylalanine residue in N348P and S454P. position 489 With either glycine or alanine. [0118] In R. marinas isoamylase (SEQ ID NO 3): [0109] Engineering for improved thermostability [0119] Positions for proline substitution include G154P, [0110] When engineering for improved thermostability, N305P and N669P, and preferably R588P and K480P. either or both of the ?rst and second parent enZymes may be an isoamylase or a pullulanase. For obtaining improved [0120] Pullulanases: thermostability of isoamylases and pullulanases, We can focus especially on the B domain, Which has been shoWn to [0121] In B. acidopullulyticus pullulanase (SEQ ID NO be important for stability, using sequence homology infor 1): mation as further described beloW. [0122] Preferred positions include A210P, V215P, L249P, [0111] Thermostability—sequence homology K383P, S509P, T811P and G823P. [0112] Several different approaches may be used for the [0123] in B. derami?cans pullulanase (SEQ ID NO 2): purpose of obtaining increased thermostability, including proline substitutions, Gly to Ala substitutions and Asn and [0124] Preferred positions include G306P, V311P, L345P, Gln substitutions. Further details and examples of these D605P, T907P and A919P. approaches are provided beloW. [0125] Gly to Ala substitutions: for example: [0113] Proline substitutions: [0126] In R amyloderamosa isoamylase (SEQ ID NO 4): [0114] Proline substitutions, i.e. replacing one or more non-proline amino acid residues With a proline residue, are [0127] 6181A suggested as an approach for obtaining thermostability on [0128] Asn (N) and Gln (Q) substitutions: the basis of sequence alignment of isomylases and pullula nases. Examples of possible proline substitutions are pro [0129] The neW residues are chosen from all 20 possible vided in the folloWing. amino acid residues, but preferably residues in a homolo gous position as seen from sequence alignment, Leu, Ile, [0115] Isoamylases: Phe, Ala, Thr, Ser and Tyr being preferred. Of special [0116] In R amyloderamosa isomylase (SEQ ID NO 4): interest are the folloWing:

SEQ ID NO 1:

Loopl: N379, N384

Loop2: N426, Q432, N434 , N437 , N444, N446

Loop3: N486, N490, Q502 , N512 , N515, N521 Loop4:

Loop5: Q596

Loop6: N616, N621, Q628

Loop7: N679, N681, Q684

Loop8: N720, N722, N731 , Q732

SEQ ID NO 2:

Loopl: N475, N480

Loop2: N522, N533, N590

Loop3: N582, N608, N611 , N617 Loop4:

Loop5: Q691, Q698

Loop6: N712, N717

Loop7: N764, N775

Loop8: N815, N817, N820 US 2002/0081670 A1 Jun. 27, 2002

-continued

SEQ ID NO 3:

Loopl: —

Loop2: N227, N232

Loop3: N286, N305, N314 , N315 , N327, N333

Loop4: —

Loop5: Q405

Loop6: —

Loop7: N482, N485, N489 , N496 , N500, Q513

Loop8: N54 , N548 , N549, Q553, N555 , N560 , Q562

SEQ ID NO 4:

Loopl: Q218, Q225

Loop2: Q254, Q257, N258 , N261 , N266, N270, Q271 , N272 , N280

Loop3: N322, N348, N358 , Q359 , N364, N370, N372 , N375

Loop4: N408, N412, N421 , N424 , N428

Loop5: N468, Q471, Q477

Loop6: —

Loop7: N547, N550, N551 , Q553 , N567, Q572

Loop8: Q615, N617, N618 , N619 , N622

[0130] Modi?cations in loops 2 and 3 are of particular [0134] Activity, sequence homology and overall beta interest With regard to improving thermostability. Loop 2 is strand, alpha-helix and loop placement in sequence knoWl of interest due to its interactions With another domain in the edge N-terminal part of the sequence. Loop 3 is of interest due to [0135] Activity, either speci?c activity or speci?city, can possible association With a calcium located be transferred to pullulanases, using sequence information betWeen domain A and domain B. from eg R amyloderamosa isoamylase (SEQ ID NO: 4) [0131] Engineering for altered substrate speci?city (high isoamylase activity). Also activity, either speci?c activity or speci?city, can be transferred to isoamylases, [0132] When engineering for altered substrate speci?city, using sequence information from eg B. acidopullulyticus either or both of the ?rst and second parent enZymes may be pullulanase(SEQ ID NO: 1). The loops are analysed for an isoamylase or a pullulanase, although it is of particular speci?c residues present especially in the beginning of the interest for purposes of the present invention to obtain loop sequence, from the end of the beta-strand in isoamy improved speci?city of pullulanases toWards higher molecu lases (or suggested beta-strand in pullulanases). lar Weight branched starchy material such as glycogen and amylopectin, in other Words a transfer of “isoamylase-like” [0136] The suggested changes exempli?ed beloW apply to speci?city to a pullulanase, eg by means of modi?cations all pullulanases in the homologous positions corresponding in the loops 1-8, preferably loops 1, 2, 4 and 5. to those of the tWo pullulanases discussed: [0133] For the transfer of isoamylase-like activity to pul [0137] Providing pullulanase With isoamylase-like activ lulanase, a loop transfer from an isoamylase to a pullulanase ity: is of particular interest, for eXample by inserting loop 5 from [0138] This may be provided by substitutions in loop an isoamylase into the site for loop 5 of a pullulanase, or by regions folloWing the beta-strands in B. acidopullulyticus inserting loop 1 from an isoamylase into the site for loop 1 (SEQ ID NO 1) and B. derami?cans (SEQ ID NO 2) of a pullulanase With the numbering indicated in Table 1. pullulanase:

After strand: Beta-1 Beta-2 Beta-3 Beta-4 Beta-5 Beta-6 Beta-7 Beta-8

B. acido. D137G N437Y F489G,A M555A 6581A I614Y,F N668G E711D Seq. ID. No.1 T585A,D W672EKQA US 2002/0081670 A1 Jun. 27, 2002

[0139] For transfer of the high activity of R amyloa'era [0142] Site-directed mutagenesis mosa isoamylase towards higher molecular Weight branched [0143] Once an isoamylase or pullulanase encoding DNA starchy material to R. marinus isoamylase or other isoamy sequence has been isolated, and desirable sites for mutation lases, or to pullulanases, a sequence alignment is performed identi?ed, mutations may be introduced using synthetic as described above. By assessing sequence homology and oligonucleotides. These oligonucleotides contain nucleotide taking into consideration the “structure” of the enZymes as sequences ?anking the desired mutation sites. In a speci?c described above, strategies for mutation can be deduced. method, a single-stranded gap of DNA, the enZyme-encod [0140] The transfer of higher activity from P. amyloa'era ing sequence, is created in a vector carrying the enZyme mosa is preferably performed Without losing the thermosta gene. Then the synthetic nucleotide, bearing the desired bility of R. marinus isoamylase in any substantial degree. mutation, is annealed to a homologous portion of the single Although it may generally be dif?cult to alter substrate stranded DNA. The remaining gap is then ?lled in With DNA activity Without altering thermostability, it is contemplated polymerase I (KlenoW fragment) and the construct is ligated that the present invention Will alloW the obtainment of a using T4 . A speci?c eXample of this method is higher activity While at the same time substantially main described in Morinaga et al., (1984), Biotechnology 2, p. taining the high thermostability in R. marinus isoamylase as 646-639. U.S. Pat. No. 4,760,025 discloses the introduction Well as in the more thermostable pullulanases. This is made of oligonucleotides encoding multiple mutations by per possible by aligning isoamylases and pullulanases to be forming minor alterations of the cassette. HoWever, an even mutated With the “key alignment” and selecting parent greater variety of mutations can be introduced at any one enZymes to be mutated as Well as speci?c amino acid time by the Morinaga method, because a multitude of residues and regions to be mutated using information oligonucleotides, of various lengths, can be introduced. obtained from such alignments of amino acid sequences. [0144] Another method for introducing mutations into [0141] The list beloW provides examples of possible muta enZyme-encoding DNA sequences is described in Nelson tions, based on these principles, that may be performed to and Long, (1989), Analytical Biochemistry 180, p. 147-151. obtain higher activity of R. marinus (SEQ ID NO 3) toWards It involves the 3-step generation of a PCR fragment con the higher molecular Weight starchy materials. taining the desired mutation introduced by using a chemi

Mutations for higher activity of SEQ ID NO 3:

Loopl: K183E, L184Q, H185D, P186T, E1875, V188T, E190A,

Preferred; L184Q, P186T, E1875, Pl9lQ

Loop2: H222Q, A223E, K224T, V225Q, H226N, R228A, 1122911, L230D, insert VPN between 231 and 232 , E2325, R233D, G234A, 1323511,

R236Q, 1124211, P243T, L244E, C245N, A2485, E250D, P251R

Preferred; K224T, V225Q, R228A, P251R

Loop3: G289A, V293T, L294W, insertion of T55DPTT between 294 and

295, G295A, P296T, T2971, L298Y, P300W, 130313, R306A, A307T,

K310E, A3llL, D3l2T, P3135, N314G, delete P316, R317Q,

L319F, V320Y, 1132211, T3251, N327A, T328N, L329F, 1333011, V33lT,

Preferred; P296T, R306A, P3135, delete P316 , V33lT, P334T

Loop4: A4045, A405V, A407G

Loop5: D397A, V3981, P400G, G401N, G4025, V405L, H407G,

Q4llG